Lipids are small molecules with large structural and chemical diversity. Over the past two decades, technologies such as chromatography and mass spectrometry have driven the biochemical analyses of complex lipid mixtures, tremendously advancing our knowledge of lipid diversity. Lipid extraction (preceding analysis), however, is still largely based on partitioning procedures developed in the 1950s. Although appropriate for many abundant components, these approaches result in variable recovery of the less-abundant and highly charged lipids, including phosphorylated signaling lipids.
Phosphate is common amongst biological lipids and present in either monoester and/or diester configurations, with most biologically active lipids containing at least one phospho-monoester. Sphingosine-1-phosphate (S1P) is an example of such a biologically active lipid. Long-chain amino alcohols, generally referred to as long-chain bases or LCBs (Pruett, S. T. et al. J Lipid Res 49, 1621-1639 (2008)), and their phosphorylated forms (LCB-P) display particularly diverse chemistries across biological species and tissues. In human plasma, the most abundant LCB-P is S1P, the phosphorylated derivative of [(2S, 3R, 4E)-2-aminooctadec-4-ene-1,3-diol], which has the structure:

S1P is currently emerging as a valid biomarker for life-threatening illnesses such as multiple sclerosis, cardiovascular disease and cancer. The commercially available method to detect S1P (antibody-based ‘ELISA’ kit) has the major disadvantages that only high S1P concentrations can be analysed and that there is a relatively high risk of false results because of the cross-reactivity of the antibodies with similar molecules. There is therefore a need for improved methods of detection of S1P. In addition to the more common LCB-Ps such as S1P, animal and plant cells and tissues comprise other LCB-Ps. Little is known about the existence of other LCB-Ps of different chemical and structural composition, despite the presence of various non-phosphorylated LCB precursors. In order to investigate the structures and functions of these compounds, it is necessary to isolate, quantify and characterize them.
Animal and plant cells and tissues also comprise other mono-phosphorylated compounds such as phosphoinositides (PIPs), phosphatidic acid, lysophosphatidic acid and ceramide-1-phosphate; phosphodiester compounds, for example phosphodiester lipids; and compounds such as lipids which contain neither phosphate mono-esters nor phosphate-diesters.
There is therefore a need to isolate, quantify and characterize phosphate ester-containing compounds such as phospholipids, particularly LCB-Ps, as well as phosphoinositides, phosphatitic acid, lysophosphatidic acid and ceramide-1-phosphate in the tissues and body fluids of humans and other animal species, for example mammals; and also in plants and microorganisms.
The present inventors have developed polymers which selectively bind phosphate esters.